This proposal examines the interaction between neutral endopeptidase (NEP), the substance P receptor (SPR) and substance P (SP) in smooth muscle of the small intestine. NEP is an ectoenzyme that is expressed by intestinal muscle cells. SP, a major stimulant of contraction of intestinal muscle, is the kinetically most favorable substrate of NEP. In many systems, specific inhibitors of NEP potentiate the biological actions of SP. Therefore, NEP regulates the biological actions of SP by controlling the number of peptide molecules that are available to interact with the SPR. Despite the undoubted importance of SP in the regulation of intestinal motility, the interaction between NEP, SPR and SP in the intestine has not been thoroughly examined. this proposal aims to elucidate the physiological function of NEP by examining the interactions between NEP, SPR and SP in the small intestine. Specific aim 1 examines the distribution of NEP, SPR and SP in the intestine and studies the anatomical relationship between cells that express NEP and SPR and SP containing nerves. The cellular location of NEP and SPR protein and mRNA will be determined by immunocytochemistry and in situ hybridization histochemistry and the relative abundance of mRNA will be determined using a ribonuclease protection assay. Specific aim 2 examines the degradation of SP by homogeneous preparations of intact intestinal muscle cells and investigates the effects of selective inhibitors of NEP on SP-stimulated contraction of dispersed muscle cells and muscle strips. The effects of inhibitors on SP release will also be examined. Specific aim 3 examines the interaction of NEP, SPR and SP in the inflamed intestine. SP is a mediator of inflammation in many systems. Inflammation of the jejunum in rats infected with Trichinella spiralis is associated with an 8-fold increase in the concentration of immunoreactive SP and 12-fold decrease in the enzymatic activity of NEP in the longitudinal muscle layer. Therefore, mRNA encoding for NEP, SPR and SP will be quantified in the inflamed rat intestine using a ribonuclease protection assay and the cells expressing NEP, SPR and SP will be localized by immunohistochemistry and in situ hybridization. The functional consequences of changes in expression of NEP, SPR and SP to the regulation of intestinal motility will be examined using highly selective inhibitors of NEP and specific antagonists of the SPR. Specific aim 4 examines the interaction between NEP and SPR in clonal cell lines that either constitutively express both NEP and SPR, such as intestinal muscle cells, or which are transfected with cDNA for NEP or SPR. The consequences of transfecting cells with cDNA for NEP on the response to SP will be investigated. Knowledge of interaction between NEP, SPR and SP will provide basic information about the nature of peptidergic neurotransmission in the normal and inflamed intestine.